Claims
- 1. A Hepatitis C Virus (HCV) replicon that efficiently replicates in a eukaryotic cell, wherein the replicon comprises a nucleic acid sequence encoding a genomic fragment of HCV and a nucleic acid sequence encoding an acetyl transferase selectable marker.
- 2. The Hepatitis C Virus (HCV) replicon according to claim 1 wherein the genomic fragment of HCV encodes a fragment of the genome of an HCV of a genotype selected from the group consisting of HCV type 1, HCV type 2, HCV type 3, HCV type 4, HCV type 5 and HCV type 6.
- 3. The Hepatitis C Virus (HCV) replicon according to claim 2 wherein the genomic fragment of HCV is a genomic fragment of HCV type 1.
- 4. The Hepatitis C Virus (HCV) replicon according to claim 3 wherein the HCV type 1 is an HCV of subtype 1a.
- 5. The Hepatitis C Virus (HCV) replicon according to claim 3 wherein the HCV type 1 is an HCV of subtype 1b.
- 6. The Hepatitis C Virus (HCV) replicon according to claim 1 wherein the eukaryotic cell is a mammalian cell.
- 7. The Hepatitis C Virus (HCV) replicon according to claim 6 wherein the mammalian cell is a primate cell.
- 8. The Hepatitis C Virus (HCV) replicon according to claim 7 wherein the primate cell is a human cell.
- 9. The Hepatitis C Virus (HCV) replicon according to claim 8 wherein the human cell is a human hepatoma cell.
- 10. The Hepatitis C Virus (HCV) replicon according to claim 9 wherein the human hepatoma cell is an Huh-7 cell.
- 11. The Hepatitis C Virus (HCV) replicon according to claim 9 wherein the human hepatoma cell is a HepG2 cell.
- 12. The Hepatitis C Virus (HCV) replicon according to claim 1 wherein the eukaryotic cell is an insect cell, an amphibian oocyte or a yeast cell.
- 13. The Hepatitis C Virus (HCV) replicon according to claim 1 wherein the acetyl transferase selectable marker is a puromycin N-acetyl transferase selectable marker.
- 14. The Hepatitis C Virus (HCV) replicon according to claim 1 wherein the genomic fragment of HCV encodes nonstructural proteins and does not encode E1, E2, or p7 structural proteins.
- 15. The Hepatitis C Virus (HCV) replicon according to claim 14 wherein the nonstructural proteins comprise NS3, NS4A, NS4B, NS5A and NS5B.
- 16. The Hepatitis C Virus (HCV) replicon according to claim 1 wherein the genomic fragment of HCV encodes nonstructural proteins and also encodes one or more HCV structural proteins.
- 17. The Hepatitis C Virus (HCV) replicon according to claim 16, which has the characteristics of HCV1a/pac.
- 18. The Hepatitis C Virus (HCV) replicon according to claim 17 which is HCV1a/pac.
- 19. The Hepatitis C Virus (HCV) replicon according to claim 15 wherein the nonstructural proteins comprise NS2, NS3, NS4A, NS4B, NS5A and NS5B.
- 20. A eukaryotic cell line comprising a Hepatitis C Virus (HCV) replicon that efficiently replicates in the eukaryotic cell, wherein the replicon comprises a nucleic acid sequence encoding a genomic fragment of HCV and a nucleic acid sequence encoding an acetyl transferase selectable marker.
- 21. The eukaryotic cell line according to claim 20 wherein the eukaryotic cell is a mammalian cell.
- 22. The eukaryotic cell line according to claim 21 wherein the mammalian cell is a primate cell.
- 23. The eukaryotic cell line according to claim 22 wherein the primate cell is a human cell.
- 24. The eukaryotic cell line according to claim 23 wherein the human cell is a human hepatoma cell.
- 25. The eukaryotic cell line according to claim 24 wherein the human hepatoma cell is a Huh-7 cell.
- 26. The eukaryotic cell line according to claim 25 wherein the human hepatoma cell is a HepG2 cell.
- 27. A Hepatitis C Virus (HCV) type 1a replicon that efficiently replicates in a eukaryotic cell, wherein the replicon comprises a nucleic acid sequence encoding a genomic fragment of HCV type 1a and a nucleic acid sequence encoding an acetyl transferase selectable marker.
- 28. The Hepatitis C Virus (HCV) type 1a replicon according to claim 27 wherein the eukaryotic cell is a mammalian cell.
- 29. The Hepatitis C Virus (HCV) type 1a replicon according to claim 28 wherein the mammalian cell is a primate cell.
- 30. The Hepatitis C Virus (HCV) type 1a replicon according to claim 29 wherein the primate cell is a human cell.
- 31. The Hepatitis C Virus (HCV) type 1a replicon according to claim 30 wherein the human cell is a human hepatoma cell.
- 32. The Hepatitis C Virus (HCV) type 1a replicon according to claim 31 wherein the human hepatoma cell is an Huh-7 cell.
- 33. The Hepatitis C Virus (HCV) type 1a replicon according to claim 31 wherein the human hepatoma cell is a HepG2 cell.
- 34. The Hepatitis C Virus (HCV) type 1a replicon according to claim 27 wherein the eukaryotic cell is an insect cell, an amphibian oocyte or a yeast cell.
- 35. The Hepatitis C Virus (HCV) type 1a replicon according to claim 27 wherein the genomic fragment of HCV type 1a encodes nonstructural proteins and does not encode E1, E2, or p7 structural proteins.
- 36. The Hepatitis C Virus (HCV) type 1a replicon according to claim 35 wherein the nonstructural proteins comprise NS3, NS4A, NS4B, NS5A and NS5B.
- 37. The Hepatitis C Virus (HCV) type 1a replicon according to claim 27 wherein the genomic fragment of HCV type 1a encodes nonstructural proteins and also encodes one or more HCV type 1 a structural proteins.
- 38. The Hepatitis C Virus (HCV) type 1a replicon according to claim 36 wherein the nonstructural proteins comprise NS2, NS3, NS4A, NS4B, NS5A and NS5B.
- 39. The Hepatitis C Virus (HCV) type 1a replicon according to claim 27 wherein the selectable marker is a drug resistance marker.
- 40. A eukaryotic cell line comprising a Hepatitis C Virus (HCV) type 1a replicon that efficiently replicates in the eukaryotic cell, wherein the replicon comprises a nucleic acid sequence encoding a genomic fragment of HCV type 1a and a nucleic acid sequence encoding a acetyl transferase selectable marker.
- 41. The eukaryotic cell line according to claim 40 wherein the eukaryotic cell is a mammalian cell.
- 42. The eukaryotic cell line according to claim 41 wherein the mammalian cell is a primate cell.
- 43. The eukaryotic cell line according to claim 42 wherein the primate cell is a human cell.
- 44. The eukaryotic cell line according to claim 43 wherein the human cell is a human hepatoma cell.
- 45. The eukaryotic cell line according to claim 44 wherein the human hepatoma cell is a Huh-7 cell.
- 46. The eukaryotic cell line according to claim 44 wherein the human hepatoma cell is a HepG2 cell.
- 47. A screening method for identifying a compound that inhibits the propagation of Hepatitis C Virus (HCV) comprising:
(a) providing a cell line comprising an HCV replicon that efficiently replicates in the eukaryotic cell, wherein the replicon comprises a nucleic acid sequence encoding a genomic fragment of HCV and a nucleic acid sequence encoding an acetyl transferase selectable marker; (b) incubating the cell line with the compound in a growth medium that selects for the acetyl transferase selectable marker under suitable conditions for growth of the cell line and assessing the growth of the cell line; (c ) providing an isogenic cell line comprising a replicon that efficiently replicates in the cell wherein the replicon comprises a replication origin that is not an HCV replication origin and a nucleic acid sequence encoding the acetyl transferase selectable marker, or an isogenic cell line comprising a nucleic acid sequence encoding the acetyl transferase selectable marker wherein the replicon does not comprise any HCV nucleic acid sequences; (d) incubating the isogenic cell line with the compound in a growth medium that selects for the acetyl transferase selectable marker under suitable conditions for growth of the isogenic cell line and assessing the growth of the isogenic cell line; and (e) comparing the growth assessed in (b) with the growth assessed in (d), wherein when the growth assessed in (b) is less than the growth assessed in (d), the compound is identified as a compound that inhibits the propagation of the HCV.
- 48. A screening method for identifying a compound that inhibits the propagation of Hepatitis C Virus (HCV) type 1 a comprising:
(a) providing a cell line comprising an HCV type 1a replicon that efficiently replicates in the eukaryotic cell, wherein the replicon comprises a nucleic acid sequence encoding a genomic fragment of HCV type 1a and a nucleic acid sequence encoding a acetyl transferase selectable marker; (b) incubating the cell line with the compound in a growth medium that selects for the selectable marker under suitable conditions for growth of the cell line and assessing the growth of the cell line; (c ) providing an isogenic cell line comprising a replicon that efficiently replicates in the cell wherein the replicon comprises a replication origin that is not an HCV replication origin and a nucleic acid sequence encoding the acetyl transferase selectable marker, or an isogenic cell line comprising a nucleic acid sequence encoding the acetyl transferase selectable marker wherein the replicon does not comprise any HCV nucleic acid sequences; (d) incubating the isogenic cell line with the compound in a growth medium that selects for the selectable marker under suitable conditions for growth of the isogenic cell line and assessing the growth of the isogenic cell line; and (e) comparing the growth assessed in (b) with the growth assessed in (d), wherein when the growth assessed in (b) is less than the growth assessed in (d), the compound is identified as a compound that inhibits the propagation of HCV subtype 1a.
- 49. A compound that inhibits the propagation of Hepatitis C Virus (HCV) identified by the screening method of claim 47 or claim 48.
- 50. A process for making a pharmaceutical compound useful for treating a Hepatitis C Virus infection, the process comprising:
(a) providing candidate pharmaceutical compounds, (b) screening the candidate pharmaceutical compounds in accordance with claim 47 or 48, (c) preparing the identified candidate pharmaceutical compound manufactured under Good Laboratory Practice (GLP) conditions.
- 51. A process for making a pharmaceutical compound useful for treating a Hepatitis C Virus infection according to claim 50, wherein the identified candidate pharmaceutical compound provided is manufactured according to Good Manufacturing Practice (GMP) conditions.
- 52. A process for making a pharmaceutical compound useful for treating a Hepatitis C Virus infection according to claim 50, wherein the identified candidate pharmaceutical compound provided is of clinical grade.
Government Interests
[0001] This work was supported in part by grant awards R37 A115122 and R01 A132100 from the National Institutes of Health. The government may have certain rights in this invention.